The present invention is in the field of enzymatic cleavage (deacylation), especially one-step cleavage of the 7-aminoadipolyl side chain (also referred to as 7-.alpha.-aminoadipyl) of cephalosporin C. Since the 7-aminoadipolyl side chain is removed by cleavage of an amide linkage, the particular enzyme which accomplishes the conversion is referred to herein as an amidase. Cephalosporin C itself is a fermentation product which is the starting point for nearly all currently marketed cephalosporins. However, synthetic manipulation to produce these various commercial cephalosporins basically starts with the 7-aminocephalosporanic acid, which must be derived from the cephalosporin C by cleavage of the 7-aminoadipolyl side chain.
Currently, the method of choice in the art for cleaving the 7-aminoadipoyl side chain is chemical. The basic imino-halide process requires blocking of the amino and carboxyl groups on the 7-aminoadipolyl side chain, and several methods for accomplishing this are currently used. However, as presently employed, the chemical cleavage process has serious disadvantages. Among these are the requirements of a multi-step and complex process, extremely low operating temperatures, expensive reagents, significant quantities of process by-products resulting in effluent treatment problems, and purification of a highly impure starting material before chemical treatment beings. Consequently, there has been an ongoing search for a microbiological or fermentative process which would achieve enzymatic deacylation of cephalosporin C to provide 7-aminocephalosporanic acid on a more economic basis than the chemical process currently in use.
However, this search for a successful microbiological process has largely proved futile, certainly with respect to one of commercial scale. This is a result of the particular characteristics of the aminoadipoly side chain of the cephalosporin C molecule, since, by contrast, penicillin G, which has a phenylacetyl side chain, has been successfully deacylated by enzymatic cleavage using penicillin acylase produced by a variety of microorganisms. Reports of successful one-step enzymatic deacylation of cephalosporin C in the literature, on the other hand, are often unreproducible or provide only very marginal yields.
Moreover, no person to date has succeeded in isolating and sequencing an enzyme from the genus Bacillus, cephalosporin C amidase, which can achieve one-step cleavage of the aminoadipoly side chain of cephalosporin C. Nor, has anyone isolated and sequenced the gene which encodes the cephalosporin C amidase enzyme, or succeeded in expressing that gene in a prokaryotic host.
A summary of the literature which describes these ongoing efforts to achieve enzymatic cleavage of cephalosporin C is set out below.
______________________________________ 1. One-Step Enzymatic Deacylation: Ceph C .fwdarw. 7-ACA Dev. Ind. Microbial., 5, 349 (1964) U.S. Pat. No. 3,239,394 (Merck) Soil enrichment method Achromobacter, of screening and selecting Brevibacterium for microorganisms Flavobacterium Jap. Pat. Pub. 53-94093 (1978) (Meiji) Pseudomonas sp. BN-188 Jap. Pat. Pub. 52-143289 (1977) Aspergillus sp. U.S. Pat. No. 4,141,790 Alternaria sp. (Meiji) U.S. Pat. No. 4,774,179 (1988) Pseudomonas sp. Jap. Pat. Pub. 61-21097 (1986) SE-83 and (Asahi) SE-495 Fr. Pat. 2,241,557 (1975) Bacillus cereus (Aries) var. fluorescens Jap. Pat. Pub. 52-082791 (1977) Bacillus (Toyo Jozo) NRRL B 5385 megaterium N-(N'-phenylthiocarbamyl)- cephalosporin C 7-ACA Ger. Pat. 3,447,023 (1986) Bacillus (Hoechst) licheniformis In the presence of a-keto acids; enzyme is D-amino acid transaminase 2. One-Step Enzymatic Deacylation: Penicillin G .fwdarw. 6-APA Jap. Pat. Pub. 58-190399 (1983) Bacillus (Shionogi) megaterium var. penicilliticum ATCC 14945 U.S. Pat. No. 3,144,395 (1964) Bacillus (Olin Mathieson) megaterium Br. Pat. Pub. 2,142,336A (1985) Bacillus (Squibb) megaterium 3. Two-Step Enzymatic Deacylation: Ceph C .fwdarw. 7-ACA U.S. Pat. No. 3,960,662 (1976) Agric. Biol. Chem. 45, 1561-67 (1981) (Toyo Jozo) Deamination with D-amino Pseudomonos sp. acid oxidase followed by deacylation EP-A- 0 275 901-A2 (1988) (Hoechst) i) Ceph C GL-7-ACA* Trigonopsis variabilis [U.S. Pat. No. 3,801,458 (1974) (Glaxo)] ii) GL-7-ACA* 7-ACA Pseudomonas gamma-glutamyl- Anthrobacter transpeptidase parafineus Bacillus subtilis 4. Enzymatic Deacylation: GL-7-ACA* .fwdarw. 7-ACA Jap. Pat. Pub. 52-128293 (1977) Bacillus, 53-86094 (1978) Arthrobacter, (Banyu) Alcaligenes 5. Enzymatic Deacylation: Other .fwdarw. 7-ACA a) Phenoxy- and Phenylacetyl 7-ADCA 7-ADCA U.S. Pat. No. 3,821,081 (1974) Bacillus megaterium Process Biochem., 11, 21 (1976) (Toyo Jozo) U.S. Pat. No. 3,749,641 (1973) 61 different (Takeda) genera U.S. Pat. No. 3,915,798 (1975) Anthrobacter simplex Belg. Pat. No. 780,676 Kluyvera citrophila (Toyo Jozo) Proteus rettgeri Bacillus megaterium b) Phenoxy-7-ADCA .fwdarw. 7-ADCA U.S. Pat. No. 3,880,713 (1975) Erwinia (Glaxo) aroideae c) Cephalothin .fwdarw. 7-ACA U.S. Pat. No. 3,522,250 (1970) Escherichia (American Home Products) coli d) Various cephalosporins .fwdarw. 7-ACA U.S. Pat. No. 3,930,949 (1976) E. coli (Bayer) penicillin acylase U.S. Pat. No. 3,962,036 (1976) E. coli, (Ciba-Geigy) Bacillus megaterium, subtilis, 3-lower alkoxy-7-acyl Micrococcus cephalosporins; roseus microorganisms lysodeikticus possessing acylase activity Alcaligenes faecalis, Aerobacter cloacae, Fusarium avenaceum, semitectum, Emericellopsis minima, Pennicillium chrysogenum, Aspergillus ochraceus, Trichophyton mentagrophytes, Epidermophyton floccosum Streptomyces lavendulae Jap. Pat. Pub. 50-107186 (1975) Arthrobacter, (Toyo Brewing) Bacillus, Escherichia, phenylacetamido 7-ACA Kluyvera, derivatives are deacylated Micrococcus, Nocardia, Proteus, Xanthomonas, 6. Enzymatic Acylation: 7-ACA .fwdarw. Other U.S. Pat. No. 3,945,888 (1976) E. coli, (Takeda) Bacillus, Proteus, 7-ACA .fwdarw. cephalosporins Pseudomonas Jap. Pat. Pub. 54-110394 (Banyu) 7-ACA .fwdarw. cephapirin Arthrobacter viscosus 7. One-Step/Two Enzyme Deacylation: Ceph C .fwdarw. 7-ACA Jap. Pat. Pub. 63 74,488 (1988) Trigonopsis variabilis, (Asahi) Comamonas Recombinant E. coli expression of D-amino acid oxidase and GL-7-ACA* acylase construct ______________________________________ *GL-7-ACA = glutaryl 7ACA = 3acetoxymethyl-7-beta-(4-carboxybutanamido)ceph-3-em-4-carboxylic acid